PROJECT SUMMARY Neurons in the brain have limited energy reserves and thus rely on a ?just-in-time? delivery strategy in which active neurons signal to the brain microvasculature to increase regional cerebral blood flow (CBF), resupplying nutrients and oxygen as well as removing toxic metabolites. Despite extensive study, the mechanisms underlying the functional linkage between neuronal metabolic demand and vascular supply, termed neurovascular coupling (NVC), remain poorly understood. Blood flow to the brain is mediated by parenchymal arterioles and hundreds of miles of capillaries, which enormously extend the territory of perfusion. We recently presented evidence supporting the concept that brain capillaries act as a neuronal activity-sensing network, demonstrating that brain capillary endothelial cells (cECs) are capable of initiating an electrical (hyperpolarizing) signal in response to neuronal activity that propagates upstream to cause dilation of feeding arterioles and increase blood flow locally at the site of signal initiation. We have established the mechanistic basis for this electrical signal, showing that neuron- and/or astrocyte-derived potassium (K+) is the critical mediator and identifying the strong inward rectifier K+ channel, Kir2.1, as the key molecular player. We have recently discovered that a second fundamental NVC mechanism based on calcium (Ca2+) signaling, with distinct kinetics and regulatory features, also operates in brain capillaries, and can be initiated by the putative NVC mediator prostaglandin E2 (PGE2). We have further found that a mechanism initiated by Gq-protein coupled receptor signaling and mediated by dynamic changes in membrane phosphatidylinositol 4,5- bisphosphate (PIP2) levels controls the balance between electrical and Ca signaling. Additional preliminary 2+ data support a role for gasotransmission via Ca2+-dependent endothelial nitric oxide signaling and pericyte- mediated regulation of capillary blood flow in modulating NVC. The immediate goals of this proposal are to create an integrated view of electrical, Ca2+ and related regulatory signaling mechanisms at molecular, biophysical, and computational-modeling levels by examining their operation in increasingly complex segments of the brain vasculature ex vivo, in vivo, and in silico. Ultimately, we propose to weave these research threads together to create a systems-level view of physiological capillary-to-arteriole/pial artery signaling in the brain, and test the concept that gradual degradation of this sensory web and the attendant progressive decay of cerebrovascular function contributes to small vessel diseases of the brain.